| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
*
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599;
Department of Pathology, University of Florida, Gainesville, FL 32610; and
The Jackson Laboratory, Bar Harbor, ME 04609
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cell-specific Th2 cells in regulating insulin-dependent
diabetes (IDDM). Whether a homogeneous population of Th2 cells having a
defined cell Ag specificity can prevent or suppress autoimmune
diabetes is still unclear. In fact, recent studies have demonstrated
that cell-specific Th2 cell clones can induce IDDM. In this study
we have established Th cell clones specific for glutamic acid
decarboxylase 65 (GAD65), a known cell autoantigen, from young
unimmunized nonobese diabetic (NOD) mice. Adoptive transfer of a
GAD65-specific Th2 cell clone (characterized by the secretion of IL-4,
IL-5, and IL-10, but not IFN-
or TGF-) into 2- or 12-wk-old NOD
female recipients prevented the progression of insulitis and subsequent
development of overt IDDM. This prevention was marked by the
establishment of a Th2-like cytokine profile in response to a panel of
cell autoantigens in cultures established from the spleen and
pancreatic lymph nodes of recipient mice. The immunoregulatory function
of a given Th cell clone was dependent on the relative levels of
IFN- vs IL-4 and IL-10 secreted. These results provide direct
evidence that cell-specific Th2 cells can indeed prevent and
suppress autoimmune diabetes in NOD mice. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cells found in the islets of Langerhans located
in the pancreas. Both cell-specific Ab and T cell responses can be
detected in persons with IDDM or at increased risk for the disease and
in spontaneous animal models for IDDM such as the nonobese diabetic
(NOD) mouse (1, 2, 3). Studies in the NOD mouse have
established that the primary mediators of cell destruction are
CD4+ and CD8+ T cells (for
review, see Ref. 4). Currently, the events that lead to
the breakdown of self tolerance within the T cell compartment and
subsequent progression of the diabetogenic response remain largely ill
defined. However, several studies suggest that cell-specific
autoimmunity is in part the result of defective peripheral
immunoregulation (3, 4, 5). Indeed, pathogenic
cell-specific CD4+ T cells typically exhibit a
Th1 cell phenotype, and injection of cytokines that promote Th1 cell
development and effector function exacerbate disease in NOD mice
(6).
Conversely, CD4+ Th2 cells that inhibit Th1 cell
differentiation through the secretion of IL-4 and IL-10 (7, 8) are believed to have a regulatory role in IDDM
(3, 4, 5). This hypothesis is supported by studies in the NOD
mouse that have demonstrated that cell autoimmunity is prevented or
suppressed following induction of Th2 cell reactivity by a variety of
means, including 1) systemic administration of IL-4 (9, 10), 2) transgene mediated secretion of IL-4 by cells
(11, 12), and 3) immunization with specific cell
autoantigens such as insulin (13). Furthermore, analysis
of cytokine RNA from the pancreas demonstrated that IL-4 expression at
the onset of islet infiltration corresponds to a nondestructive type of
insulitis found predominantly in NOD male mice, which typically develop
overt diabetes at a lower frequency than females (14). In
contrast, the breakdown of immunoregulation involving cell-specific
Th1 cells is thought to be partly due to a defect in efficient
induction of Th2 cells (9, 15).
Evidence demonstrating that a homogeneous population of Th2 cells with
a defined cell Ag specificity can immunoregulate IDDM is currently
lacking. For example, in studies where protection has been induced in
NOD mice, the population of Th2 effector cells is typically
heterogeneous in terms of cell Ag specificity and may consist of
other types of regulatory Th cells. Similar caveats exist for studies
demonstrating that diabetes can be prevented in young NOD mice upon
adoptive transfer of short term Th2 cell lines established from
immunized animals. Finally, the role of Th2 cells in IDDM has been
brought into question by observations that 1) disease progression is
not affected in NOD mice lacking a functional IL-4 gene (16, 17); and 2) IDDM is exacerbated in young NOD mice receiving
cell-specific Th2 cell clones established from NOD mice transgenic for
the BDC2.5 clonotypic TCR (18).
In the present study, we have investigated the potential
immunoregulatory function of cell-specific Th2 cells in autoimmune
diabetes through the use of a panel of Th cell clones specific for
glutamic acid decarboxylase 65 (GAD65). GAD65 is among the first
cell autoantigens targeted in the NOD mouse and is believed to have a
key, albeit undefined, role in both murine and human IDDM
(19, 20, 21, 22, 23, 24). Furthermore, cell autoimmunity is prevented
or suppressed in NOD mice immunized at various ages with either GAD65
protein or peptides (17, 19, 20, 25, 26). The protection
observed in these studies correlated with the induction of
GAD65-specific Th2 cell reactivity. Here we provide evidence that
adoptive transfer of a GAD65-specific Th2 cell clone established from
young unimmunized NOD mice can effectively prevent and/or suppress
established cell autoimmunity in recipient mice.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
NOD/Lt and NOD.scid mice were housed and bred under
specific pathogen-free conditions and allowed access to NIH diet
31A (Ralston Purina, St. Louis, MO). Currently, IDDM develops in
85% of NOD/Lt female mice by 1 yr of age in our colony maintained
at University of North Carolina (Chapel Hill, NC). The establishment
and screening of NOD mice homozygous for an inactivated IL-4 gene
(NOD.IL4null) have previously been described
(17).
Assessment of diabetes and insulitis
Mice were monitored weekly for the development of glycosuria with Diastix (Ames, Elkhart, IN). Two successive positive measurements were considered diagnostic of diabetes onset. Insulitis was assessed by histology. Pancreases were prepared for histology by fixation in neutral buffered formalin followed by paraffin embedding. A minimum of five sections, 90 µm apart, were cut from each block, stained with hematoxylin and eosin, and viewed by light microscopy. A minimum of 30 islets were scored for each animal. The severity of insulitis was scored as either peri-insulitis (islets surrounded by a few lymphocytes) or intrainsulitis (lymphocytic infiltration of the islets).
Antigens
The cloning and preparation of murine cell autoantigens
GAD65, heat shock protein 60 (HSP60), and carboxypeptidase H (CPH) have
been previously described (19). Briefly, the cDNAs were
engineered to encode six histidine residues at the COOH terminus of
each protein. Recombinant proteins were expressed in baculovirus
(GAD65, CPH) and Escherichia coli (HSP60) expression systems
and were purified using an Ni2+-conjugated resin
(Qiagen, Chatsworth, CA). Each recombinant protein was further purified
by preparative SDS-PAGE, electroelution, and extensive dialysis in PBS.
Peptides were synthesized using standard F-moc chemistry on a Rainin
Symphony (Rainin Instruments, Emeryville, CA) at the peptide
synthesis facility of University of North Carolina. The purity of the
peptides was verified by reverse phase HPLC and mass spectroscopy.
Establishment of GAD65-specific CD4+ Th cell clones
GAD65-specific T cell clones were established by culturing
7.5 x 106 splenocytes from 4-wk-old
unimmunized NOD female mice in a 24-well plate in 1.5 ml of RPMI 1640,
5 x 10-5 M 2-ME, 1 mM sodium pyruvate, 1x
nonessential amino acids, 1 mM glutamine, 100 U/ml of
penicillin/streptomycin (base medium) supplemented with 1.0% NOD
serum, and 10 µg/ml of murine GAD65 for 7 days. T cells (1 x
106) harvested on a Lympholyte M gradient
(Cedarlane Laboratories, Hornby, Canada) were cultured with 5 x
106 irradiated (3000 rad) NOD splenocytes in 1.5
ml of the above medium and 10 µg/ml GAD65 in a 24-well plate. Three
days later the cultures were supplemented with base medium containing
20 U/ml of murine IL-2 (BD PharMingen, San Diego, CA) and 10% FBS and
maintained for an additional 3 days. CD4+ T cells
were purified by magnetic separation using anti-CD4 magnetic beads
(Miltenyi Biotec, Auburn, CA) as recommended by the manufacturer.
GAD65-specific CD4+ T cell clones were
established by culturing 0.5 and 1.0 T cells with 2 x
105 irradiated NOD splenocytes/well in 96-well
round-bottom plates in base medium containing 10% FBS, 20 U/ml IL-2,
and 10 µg/ml GAD65. Subclones of the parental 6H1 T cell clone were
established in an identical manner. Following expansion, the
monoclonality of a given T cell clone was determined by flow cytometric
analysis of TCR V
2 and V14 chain usage. 1F2 cells were
established in a similar manner, with the exception of using the
GAD65-specific p524–543 peptide to stimulate and propagate T cells
instead of intact GAD65. Once established, T cell clones were
maintained in culture on a 21-day growth cycle. Briefly, 2 x
106 T cell clones were stimulated with 2 x
107 irradiated (3000 rad) splenocytes and 10
µg/ml GAD65 or peptide in an upright T-25 tissue culture flask. On
day 3, base medium containing 10% FBS, 20 U/ml IL-2 plus 40 U/ml IL-4
(BD PharMingen) was added to the cultures, and T cell clones were
expanded accordingly up to 21 days.
T cell clone proliferation and cytokine assays
Th cell clone proliferation was measured by culturing in
triplicate 2.0 x 104 T cells plus 2.0
x 105 irradiated (3000 rad) splenocytes/well
(0.2 ml) in a 96-well round-bottom microtiter plate for 72 h with
the appropriate Ag. Proliferation was assessed by measuring the amount
of [3H]thymidine incorporation (1 µCi/well)
during the last 18 h of culture and was expressed as a stimulation
index (mean cpm of response to Ag divided by mean cpm with medium
only). To measure cytokine secretion, supernatants from cultures
established as described above were harvested after 48 h, pooled,
and used in a capture ELISA system. The levels of IFN-, IL-4, IL-5,
and IL-10 were determined in triplicate in 0.1 ml of supernatant. Abs
were obtained from BD PharMingen, and ELISA was conducted as
recommended by the manufacturer. Standard curves were established to
quantitate the amount of the respective cytokines in the culture
supernatants. The lower limits of detection for IFN-, IL-4, IL-5,
and IL-10 were typically 50, 25, 30, and 30 pg/ml, respectively.
Adoptive transfer of GAD65-specific T cell clones
For adoptive transfer experiments, Th cell clones were harvested on a Lympholyte M gradient 7 days after Ag stimulation, extensively washed, and resuspended in PBS. Two-week-old NOD or 4-wk-old NOD.scid female mice received two i.p. or i.v. (tail vein) injections of 107 cells (0.1 ml) within 14 days. Twelve-week-old NOD or NOD.IL4null female mice received two i.v. injections of 2 x 107 cells (0.1 ml) over 14 days. Animals were then monitored for diabetes.
In vitro cytokine assay
Lymphocyte cytokine secretion in cultures prepared from adoptive
transfer recipients in response to the panel of cell autoantigens
was determined as previously described (23). Briefly, a
splenocyte suspension was prepared from individual mice in ice cold
PBS. The splenocyte suspension was immediately centrifuged at 400
x g for 5 min at 4°C and resuspended at 2.5 x
106 cells/ml in base medium supplemented with 2%
Nutridoma-SP (Roche, Indianapolis, IN). Splenocytes (0.2 ml/well) were
incubated in 96-well flat-bottom microtiter plates in the presence of
10 µg/ml of GAD65, HSP60, or CPH or 25 µg/ml of peptide. Six wells
were used for each cell autoantigen. Culture supernatants were
harvested and pooled for each Ag treatment after 48 h, with a
capture ELISA used to measure IFN- and IL-4 in 0.1 ml of culture
supernatant (in triplicate) as described above.
Enzyme-linked immunospot (ELISPOT)
ImmunoSpot M200 plates (Cellular Technology, Cleveland,
OH) were coated overnight at 4°C with either 2 µg/ml
anti-IFN- Ab (R4-6A2, BD PharMingen) or 4 µg/ml anti-IL-4
Ab (11B11, BD PharMingen) prepared in PBS. Plates were blocked with 1%
BSA-PBS for a minimum of 2 h at room temperature and then washed
four times with PBS. Splenocytes were prepared from individual mice as
described above, with the exception of being resuspended in HL-1 medium
(BioWhittaker, Walkersville, MD). Splenocytes were then plated at
5 x 105 cells/well (0.2 ml/well).
Pancreatic lymph nodes were pooled within a given treatment group, and
the resulting suspension was prepared in HL-1 medium and plated at
2.5 x 105 cells/well with 5 x
105 cells/well irradiated (3000 rad) splenocytes
harvested from NOD.IL4null mice. Ag or peptide
was added to triplicate wells at a final concentration of 10 or 25
µg/ml, respectively. The plates were incubated for 24 h
(IFN-) or 48 h (IL-4) at 37°C in 5.5%
CO2 and then washed three times with PBS followed
by an additional three washes with 0.025% Tween 20-PBS. Biotinylated
anti-IFN- (XMG1.2, BD PharMingen) or anti-IL-4 (BVD6-24G2,
BD PharMingen) was added at 2 and 4 µg/ml, respectively, in 1%
BSA-PBS (0.1 ml/well). After overnight incubation at 4°C, plates were
washed three times with 0.025% Tween 20-PBS and incubated with
streptavidin-HRP (BD PharMingen; 1/2000) for 2 h at room
temperature. This was followed by three washes with 0.025% Tween
20-PBS and three washes with PBS only. Development solution consisted
of 0.8 ml of 3-amino-9-ethyl-carbazole (Sigma, St. Louis, MO; 20 mg
dissolved in 2.0 ml of dimethylformamide) added to 24 ml of 0.1 M
sodium acetate (pH 5.0), plus 0.12 ml of 3.0% hydrogen peroxide; 0.2
ml was added per well.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We and others have previously shown that GAD65-specific
CD4+ T cell reactivity is first detected in
4-wk-old NOD female mice (19, 20). To gain insight into
the role of these Th cells in disease progression and the peptide
epitope(s) targeted, we established GAD65-specific Th cell clones from
the spleens of unimmunized 4-wk-old NOD female mice using intact murine
GAD65. Our conditions included supplementing the cultures with IL-2,
but no effort was made to selectively promote either Th1 or Th2 cell
clones. Of the 89 GAD65-specific Th cell clones that were established,
88 were shown to be specific for a peptide epitope spanning amino acid
residues 217–236 (p217–236). A minimum of 10 distinct clonotypes were
detected based on TCR V-chain usage. The p217–236-specific Th cell
clones exhibited either a Th0 or a Th1 cell phenotype as determined by
IFN-, IL-4, IL-5, and IL-10 secretion. The remaining Th cell clone,
designated 6H1, differed from the rest in both peptide specificity and
phenotype. 6H1 cells proliferated in response to intact GAD65 or
p290–309 and secreted IL-4, IL-5, and IL-10, but not IFN-,
indicating a Th2 cell phenotype. Further cloning of the 6H1 Th2 cells
via limiting dilution resulted in 40 subclones. Thirty-nine of these,
represented by the 6H1E subclone, proliferated in response to GAD65 and
p290 and exhibited a typical Th2 cytokine secretion profile (Fig. 1
). Interestingly, one subclone,
designated 6H1L, secreted significant amounts of IFN- in addition to
IL-4, IL-5, and IL-10 (Fig. 1B). Messenger RNA encoding TGF- could
not be detected in either the 6H1 parental cells or the 6H1E and 6H1L
subclones as determined by RT-PCR.
|
, in response to the
GAD65-specific peptide p524–543 (Fig. 1
B). It is noteworthy
that 1F2 Th2 cells did not respond to intact GAD65 (Fig. 1A).
To assess the short-term in vivo function of the 6H1E, 6H1L, and 1F2 Th
cell clones, 2-wk-old NOD female mice received two i.v. injections of
107 cells over 14 days or were left untreated.
Four weeks after the last injection, spleen and pancreatic lymph node
cultures were prepared, and cytokine secretion in response to GAD65
protein or GAD65-derived peptides was measured. Cultures prepared from
the spleen or pancreatic lymph nodes of untreated NOD mice yielded a
typical Th1 cell cytokine profile in response to GAD65 or p290–309 and
only a marginal response to p524–543 (Fig. 2A). In marked contrast,
significantly reduced levels of IFN- and a concomitant increase in
IL-4 were detected in response to GAD65 by cultures prepared from 6H1E
recipient NOD mice (Fig. 2). Furthermore, enhanced IL-4 secretion in
response to p290–309 was detected in these cultures relative to that
in cultures prepared from untreated animals (Fig. 2B).
Cultures prepared from 1F2 recipient mice exhibited a modest decrease
in IFN- secretion in response to GAD65 and only limited, but
significant, IL-4 secretion in response to p524–543 (Fig. 2).
Furthermore, these cultures did not secrete IL-4 in response to whole
GAD65 (Fig. 2B), consistent with data demonstrating that the 1F2 Th2
cell clone responds to p524–543, but not to intact Ag (Fig. 1A). A distinct profile of cytokine secretion was observed
in cultures prepared from mice receiving the 6H1L Th cell clone. In
these cultures, both IFN- and IL-4 secretions were increased in
response to GAD65 and p290–309 relative to those in cultures
established from untreated animals (Fig. 2).
|
and 3). A greater frequency of 6H1E, 6H1L,
and 1F2 cells was detected in splenocyte vs pancreatic lymph node
cultures, as determined by responses to p290–309 and p524–543,
respectively. Furthermore, the frequency of IL-4-secreting Th cells in
response to p290–309 in either spleen or pancreatic lymph node
cultures was similar for the 6H1E and 6H1L Th cell clone recipients
(Fig. 3B). These frequencies of IL-4-secreting Th cells were
consistently greater than those detected in spleen and pancreatic lymph
node cultures established from recipients of the 1F2 Th cell clone in
response to p524–543 (Fig. 3B).
|
To investigate the effect of the GAD65-specific Th cell clones on
IDDM, adoptive transfer experiments were conducted in NOD female mice
exhibiting different stages of disease progression. As described above,
2-wk-old NOD female mice received two i.v. injections of
107 cells of a given clone and were subsequently
monitored for diabetes up to 35 wk of age. NOD mice at this young age
lack detectable cell-specific T cell and Ab reactivity and show no
infiltration of the pancreas. The majority of mice (12 of 15) receiving
1F2 cells developed IDDM at a frequency and time of onset
indistinguishable from untreated NOD female mice (12 of 15; Fig. 4A). In contrast, a
significant reduction in the frequency of overt diabetes was observed
in the group of mice receiving the 6H1E Th2 cell clone (2 of 15;
p < 10-3, by
2 test) relative to that in untreated animals
(Fig. 4A). Interestingly, adoptive transfer of the
6H1L cells had no significant effect on the onset and frequency (11 of
15) of diabetes in recipient mice (Fig. 4A), suggesting that
secretion of IFN- blocked the immunoregulatory function of the Th
cell clone. We also performed adoptive transfer experiments with four
p217–236 Th1 cell clones and found that the development of insulitis
was, in fact, enhanced in recipient mice (data not shown).
|
cell-specific autoimmunity. Euglycemic NOD female mice, 12 wk of age,
were used as recipients. Typically, 12-wk-old NOD female mice exhibit
maximal cell-specific T cell and Ab reactivity in addition to
significant insulitis. Mice received two i.v. injections of 2 x
107 1F2, 6H1E, or 6H1L cells over 14 days and
were monitored for overt diabetes up to 35 wk of age. The group of mice
receiving the 6H1E Th2 cell clone exhibited a significantly reduced
frequency of diabetes (1 of 10; p = 0.009, by
2 test) compared with untreated mice (9 of 12;
Fig. 4B). However, the majority of mice injected with the
6H1L (8 of 10) or 1F2 (9 of 10) Th cell clones continued to develop
diabetes (Fig. 4B).
Histological analysis of pancreases obtained from nondiabetic mice
receiving the respective Th cell clones at 2 or 12 wk of age further
confirmed the protective effect associated with the 6H1E Th2 cell
clone. Whereas the majority of islets exhibited extensive
intrainsulitis in the pancreases of mice injected with the 1F2 or 6H1L
Th cell clones, a significant number of islets remained free of
cellular infiltration in the pancreases of 6H1E cell recipient mice in
either age group tested
(Tables I and II). Strikingly, while a significant number of islets exhibited
peri- and intrainsulitis, further progression of insulitis was
suppressed in mice receiving the 6H1E Th2 cell clone at 12 wk of age;
the frequency of insulitis in 35-wk-old 6H1E cell recipient mice was
similar to that typically observed in untreated 12-wk-old NOD
female mice.
|
|
To determine the immune status of NOD mice receiving the 1F2,
6H1E, or 6H1L Th cell clones at 2 or 12 wk of age, splenocyte cultures
were prepared from recipient mice, and cytokine secretion in response
to GAD65, p290–309, or p524–543 was measured. As demonstrated in
Figs. 5A and 6A,
similar amounts of IFN- secretion in response to GAD65 and p290–309
were detected in cultures prepared from both nondiabetic and diabetic
mice left untreated or receiving the 1F2 cells. In addition, IL-4 was
not detected in response to p524–543 (Figs. 5B and
6B). As in the short term adoptive transfer experiments
described above (Figs. 2 and 3), reduced IFN- and increased IL-4
secretion in response to GAD65 and p290–309 were detected in cultures
prepared from nondiabetic 6H1E cell recipient mice at 2 wk (Fig. 5) or
12 wk (Fig. 6) of age. Furthermore, a
distinct Th1 cell cytokine profile similar to that in untreated animals
was detected in cultures prepared from the few 6H1E cell recipients
that developed overt diabetes (Figs. 5 and 6). This result suggests
that IDDM progressed in these animals due to the lack of in vivo
persistence or sufficient expansion of the 6H1E Th2 cell clone. This
contrasted with the 6H1L cell recipient mice, in which cultures
prepared from both nondiabetic and diabetic recipients displayed
enhanced IFN- and IL-4 secretion in response to GAD65 and p290–309
relative to untreated mice (Figs. 5 and 6).
|
|
The reduced frequency of insulitis observed in NOD mice receiving
the 6H1E Th2 cell clone at 2 or 12 wk of age (Tables I and II) strongly
suggested that protection was primarily mediated in the periphery as
opposed to the pancreatic islets. However, cells found infiltrating the
pancreas of nondiabetic recipient mice may have nevertheless consisted
of 6H1E Th2 cells. To investigate this issue further the 1F2, 6H1E, and
6H1L Th cell clones were adoptively transferred into
NOD.scid mice. Six weeks after the final injection of the Th
cell clones, NOD.scid recipients were assessed for insulitis
in addition to in vitro T cell reactivity in the spleen and pancreatic
lymph nodes. Histological examination of the pancreases prepared from
NOD.scid mice receiving any of the three Th cell clones
showed no detectable insulitis (Table III). This differed from
NOD.scid mice, which exhibited significant intrainsulitis
after receiving a suboptimal dose of splenocytes from diabetic NOD
donor mice (Table III). Importantly, cytokine secretion in response to
GAD65 and p290–309 was detected in both splenocyte and pancreatic
lymph node cultures prepared from NOD.scid mice receiving
the 6H1E or 6H1L Th cell clones (Fig. 7).
In agreement with results obtained from wild-type NOD recipients,
modest IL-4 secretion in response to p524–543 was detected in
splenocyte or pancreatic lymph node cultures established from
NOD.scid mice receiving the 1F2 Th2 cell clone.
|
|
Next, we determined whether cell-specific Th1 cell reactivity
was directly suppressed by the 6H1E Th2 cell clone or required
recruitment of other cell-specific regulatory Th2 cells. For this
set of experiments, we used NOD.IL4null mice,
which lack a functional IL-4 gene and consequently are unable to
generate typical Th2 effector cells (16, 17).
Twelve-week-old NOD.IL4null or wild-type NOD mice
received two injections of the 6H1E Th2 cell clone as described above
and were monitored for diabetes up to 45 wk of age. As expected, the
majority of wild-type NOD mice receiving the 6H1E Th2 cell clone
remained free of diabetes by 45 wk of age (Fig. 8A). Furthermore, a
significant reduction in IFN- and a corresponding increase in IL-4
secretion were detected in response to GAD65 and p290–309 (Fig. 8B). As described above, IFN-, but not IL-4 secretion in
response to GAD65 or p290–309 was detected in cultures prepared
from the few wild-type NOD mice that had received the 6H1E cells and
developed diabetes (Fig. 8B).
|
A). However, the majority of the 6H1E
NOD.IL4null recipients (7 of 10) eventually
developed overt diabetes. Adoptive transfer of the 1F2 Th2 cell
clone had no effect on the onset or frequency of diabetes in
NOD.IL4null recipients (data not shown).
Interestingly, enhanced secretion of both IL-4 and IFN- was detected
in response to p290–309 in cultures prepared from either nondiabetic
or diabetic NOD.IL4null recipients monitored long
term (Fig. 8C). Furthermore, this cytokine profile, which
resembles that observed with 6H1L cells, suggests that endogenous IL-4
was necessary to maintain the Th2 cell phenotype of the 6H1E cells in
vivo and, in turn, may explain why overt diabetes eventually developed
in recipient animals. We established individual Th cell clones from the
NOD.IL4null recipient mice and confirmed that
most of the transferred 6H1E cells obtained the capacity to secrete
IFN- (data not shown).
We recently demonstrated that immunizing NOD mice with the
GAD65-specific peptides p217–236 and p290–309 prepared in IFA
prevented IDDM and correlated with the induction of GAD65-specific
regulatory Th2 cells (17). This Th2 response was not
limited to GAD65, but had spread to other cell autoantigens.
However, peptide immunization of NOD.IL4null mice
mediated no protection or Th2 responses to either GAD65 or other
cell autoantigens. To determine whether a similar set of events could
be observed here, IL-4 secretion in response to p217–236 and the
candidate cell autoantigens HSP60 and CPH was measured in
splenocyte cultures prepared from NOD or
NOD.IL4null mice 4 wk after adoptive transfer of
6H1E cells. As demonstrated in Fig. 9, cultures prepared from wild-type NOD mice receiving the 6H1E cells
exhibited elevated levels of IL-4 relative to untreated NOD mice in
response to p290–309 as well as p217–236, HSP60, and CPH. In
contrast, IL-4 secretion was detected in cultures prepared from
NOD.IL4null recipients only in response to
p290–309 and not to the other cell autoantigens assayed.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cell-specific Th2 cells in
regulating IDDM is currently unclear. In part, this is due to a lack of
evidence demonstrating that a homogeneous population of Th2 cells with
a defined cell Ag specificity can prevent or suppress the
diabetogenic response. In the present study GAD65-specific Th2 cell
clones were established from unimmunized NOD mice using culture
conditions that promote in vitro expansion of Th cells that have
encountered cell autoantigen in vivo. Accordingly, the Th cell
clones established in this manner reflect GAD65-specific clonotypes
that occur spontaneously in vivo and provide evidence that
cell-specific Th2 cells normally exist in unmanipulated NOD mice.
However, the frequency of GAD65-specifiic Th2 cells is low, consistent
with our work and others demonstrating that Th1-like responses to GAD65
or other cell autoantigens predominate (19, 20, 25, 26). Indeed, efficient induction of regulatory Th2 cells in NOD
mice has often required an exogenous source of IL-4 (9, 10, 27) That cell-specific Th2 cells can indeed immunoregulate
IDDM was demonstrated by adoptive transfer of the 6H1E Th2 cell clone,
which prevented the progression of insulitis (Tables I and II) and the
development of diabetes in NOD recipient mice (Figs. 4 and 8A). The
immunoregulatory function of this GAD65-specific Th2 cell clone was
effective either before or after the establishment of cell-specific
Th1 cells. Prevention of IDDM by the Th cell clones correlated with a
Th2-like recall response to p290-309 and GAD65 (Figs. 5, 6, and 8) in
addition to other cell autoantigens (Fig. 9). Notably, this T cell
reactivity was dependent on the relative levels of IFN- vs Th2 cell
cytokines secreted by the Th cell clone.
A cytokine profile indicative of Th2 cell reactivity was detected in
splenocyte and pancreatic lymph node cultures established from
nondiabetic NOD mice monitored either short or long term following
adoptive transfer of the 6H1E Th2 cell clone (Figs. 2, 3, 5, 6, and 8).
These cultures typically exhibited reduced Th1 cell reactivity and
enhanced IL-4 secretion in response to GAD65 and p290–309. Strikingly,
a Th2-like cytokine profile was also established in
cultures prepared from 12-wk-old NOD recipients, an age at which
cell autoimmunity is well underway. The existing
anti-GAD65-specific Th1 cell reactivity observed in these cultures
presumably represented established Th1 effector cells at the time of
adoptive transfer of 6H1E cells. The immunoregulatory function of the
transferred 6H1E cells was also characterized by induction
of Th2 cell reactivity to other cell autoantigen determinants, such
as HSP60, CPH, and an additional GAD65-specific epitope, p217–236
(Fig. 9). This intermolecular determinant spread of the Th2 cell
phenotype has previously been shown to be important for IDDM prevention
in NOD mice immunized with GAD65-derived peptides (17).
The inability of the 6H1E cells to promote Th2 reactivity to other
cell autoantigens in NOD.IL4null mice probably
explains why diabetes eventually developed in the recipients. However,
in wild-type NOD recipients the recruitment of additional
cell-specific Th2 cells may have aided in maintaining the Th2 cell
phenotype of the 6H1E cells by providing an endogenous source of IL-4
in addition to amplifying immunoregulatory effects. Nevertheless, the
delay in the onset of diabetes in NOD.IL4null
recipients indicates that the frequency of 6H1E cells and the level of
IL-4 secretion were sufficient to directly regulate the diabetogenic
response shortly after transfer. The failure of the 1F2 Th2 cell clone
to prevent IDDM despite secreting higher levels of IL-4 and IL-10
compared with 6H1E cells (Fig. 1, C and E) may
reflect an inability of the T cells to persist or sufficiently expand
in vivo. This idea is consistent with the observation that 1F2 cells do
not respond to GAD65 protein in vitro (Fig. 1A).
In contrast, failure of 6H1L cells to protect NOD recipients from overt
diabetes demonstrated the importance of the relative balance between
IFN- and Th2-like cytokines in determining the immunoregulatory
function of Th cell clones (Fig. 4). In addition to secreting
equivalent levels of IL-4 and IL-10 in vitro (Fig. 1B), the
6H1L and 6H1E Th cell clones trafficked to the spleen and pancreatic
lymph nodes to the same extent upon adoptive transfer, as seen by the
frequency of IL-4-secreting Th cells responding to p290–309 (Fig. 3B). Nevertheless, NOD mice receiving the 6H1L Th cell clone
developed diabetes with a similar onset and frequency as untreated
animals (Fig. 4). The inability of the 6H1E Th2 cell clone to protect
NOD.IL4null mice long term provided additional
evidence that the relative levels of Th1 vs Th2 cell cytokines are the
key to determining the immunoregulatory efficacy of Th cell clones. In
the absence of endogenous IL-4, 6H1E cells began to secrete significant
levels of IFN- while continuing to secrete IL-4 (Fig. 8C)
and IL-10 (data not shown) in response to GAD65 and p290–309.
Consequently, the majority of recipient mice developed overt diabetes,
albeit with a delayed onset relative to untreated
NOD.IL4null mice (Fig. 8A) or animals
receiving the 1F2 Th2 cell clone (R. Tisch, unpublished observations).
This delayed onset of diabetes probably reflected the time during which
levels of IFN- secretion became sufficient to suppress the
immunoregulatory capacity of the 6H1E cells. We have established Th
cell clones from NOD.IL4null recipient mice and
confirmed that the enhanced IFN- secretion detected in response to
p290–309 was derived from 6H1E cells based on expression of the 6H1
clonotypic V2/V14 TCR (R. Tisch, unpublished observations).
Interestingly, adoptive transfer of an insulin-specific Th1 cell clone
has been reported to prevent diabetes in NOD recipient mice
(28). However, in this instance, the immunoregulatory
function of the Th1 cell clone was due to secretion of TGF-, which
is not expressed by 6H1E or 6H1L cells.
Another important aspect of the protection associated with the 6H1E Th2
cell clone was the inhibition of insulitis in recipient animals. NOD
mice receiving 6H1E cells at 2 or 12 wk of age exhibited significantly
reduced frequencies of insulitis relative to mice left untreated or
receiving the 1F2 or 6H1L Th cell clones (Tables I and II). This
observation is in agreement with a recent study demonstrating that
GAD65-specific Th cell lines established from NOD mice expressing IL-4
in cells limited the capacity of diabetogenic splenocytes to
infiltrate the pancreas of recipient animals in coadoptive experiments
(12). Based on the lack of detectable infiltration in
NOD.scid recipients (Table III) 6H1E cells do not appear to traffick to the islets. This contrasts
with our GAD65-specific Th cell clones that exhibit a classical Th1
cell phenotype, i.e., IFN-+,
IL-4-, IL-10-, and
TGF--, which do infiltrate the islets of
NOD.scid recipients (R. Tisch, unpublished results).
Together, these findings demonstrate that immunoregulation by the 6H1E
Th2 cell clone occurred largely in the periphery of recipient mice. The
pancreatic lymph nodes in which activation and regulation of
cell-specific T cells have been shown to occur (29, 30, 31)
probably provide key sites for immunoregulation by 6H1E cells. Indeed,
a low, but significant frequency of IL-4-secreting T cells was detected
in response to GAD65 and p290–309 in pancreatic lymph node cultures
prepared from NOD mice receiving 6H1E cells (Fig. 3B).
We suggest the following scenario to explain the immunoregulatory
effect of the 6H1E Th2 cell clone. Upon adoptive transfer, 6H1E cells
seed peripheral sites, including the spleen and pancreatic lymph nodes.
At these sites a low frequency of 6H1E cells sufficient to establish a
microenvironment conducive to Th2 cell development persist through
recognition and stimulation by endogenous GAD65. The secretion of IL-4
would promote Th2 while suppressing Th1 differentiation of uncommitted
cell-specific Th cell precursors. Additionally, IL-4 and IL-10
would modify the function of APCs to further limit commitment along the
Th1 pathway (31, 32). Induction of other cell-specific
Th2 cells amplifies the immunoregulatory effect, in addition to
providing a source of endogenous IL-4, which is required to maintain
the phenotype of 6H1E cells in vivo. In this manner, infiltration of
cell-specific Th1 cells is effectively inhibited. As indicated by
the 6H1L cell transfer data, secretion of IFN- inhibits efficient
Th2 cell commitment despite the presence of IL-4 and would also be
expected to enhance the responsiveness of naive Th cell precursors to
IL-12 to promote Th1 cell differentiation (33, 34).
Recent work by Poulin and Haskins has demonstrated that Th2 cell clones established from the BDC2.5 TCR transgenic mouse accelerated the onset of diabetes upon adoptive transfer into young NOD recipients (18). Currently, it is not clear why the GAD65-specific and the BDC2.5 derived Th2 cell clones prevent and promote IDDM, respectively. One obvious difference is the specificity of the respective Th2 cell clones. The identity of the autoantigen recognized by BDC2.5 Th cells has yet to be determined; however, differences in the level and anatomical presentation of the corresponding peptide epitopes may impact on a variety of in vivo T cell properties, including trafficking and effector function. A direct comparison between the GAD65-specific and BDC2.5-derived Th2 cell clones may provide further insight into the factors that govern regulatory Th2 cell function.
In summary, this study provides evidence that GAD65-specific Th2 cells exist in unmanipulated NOD mice, and that adoptive transfer of a GAD65-specific Th2 cell clone can effectively prevent and suppress IDDM. Nevertheless, it is apparent that immunoregulation of IDDM is complex, involving not only Th2 cells, but also a number of types of regulatory T and non-T cells (28, 35, 36, 37, 38, 39, 40, 41, 42). The task at hand is to determine the relative contributions and potential interactions between these different regulatory cells to gain a complete understanding of the events leading to the breakdown of self tolerance in IDDM and other T cell-mediated autoimmune diseases.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Roland Tisch, Department of Microbiology and Immunology, Mary Ellen Jones Building, Room 804, Campus Box 7290, University of North Carolina, Chapel Hill, NC 27599-7290. E-mail address: rmtisch{at}med.unc.edu
3 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; NOD, nonobese diabetic; CPH, carboxypeptidase H; ELISPOT, enzyme-linked immunospot; GAD65, glutamic acid decarboxylase 65; hsp60, heat shock protein 60.
Received for publication February 7, 2001. Accepted for publication March 22, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
transcription. J. Clin. Invest. 95:628.
cell autoimmunity by plasmid DNA vaccination. J. Immunol. 166:2122. production is cytokine dependent. Proc. Natl. Acad. Sci. USA 94:3189.
T cells that prevent murine insulin dependent diabetes. J. Exp. Med. 184:2167./ T cell receptor (TCR)+CD4+CD8- (NKT) thymocytes prevent insulin dependent diabetes mellitus in nonobese diabetic (NOD)/Lt mice by the influence of interleukin (IL)-4 and/or IL-10. J. Exp. Med. 187:1047.+ CD4+ thymocytes expressing L-selectin mediate "active tolerance" in the nonobese diabetic mouse. J. Immunol. 161:2620. and interleukin 4 in the prevention of autoimmune thyroiditis in rats by peripheral CD4+CD45RC- cells and CD4+CD8- thymocytes. J. Exp. Med. 189:279.This article has been cited by other articles:
|
|
 |
|
  M. J. Perone, S. Bertera, W. J. Shufesky, S. J. Divito, A. Montecalvo, A. R. Mathers, A. T. Larregina, M. Pang, N. Seth, K. W. Wucherpfennig, et al. Suppression of Autoimmune Diabetes by Soluble Galectin-1 J. Immunol., March 1, 2009; 182(5): 2641 - 2653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Li, B. Wang, J. A. Frelinger, and R. Tisch T-Cell Promiscuity in Autoimmune Diabetes Diabetes, August 1, 2008; 57(8): 2099 - 2106. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Chen, G. Han, J. Wang, R. Wang, R. Xu, B. Shen, J. Qian, and Y. Li Induction of Active Tolerance and Involvement of CD1d-Restricted Natural Killer T Cells in Anti-CD3 F(ab')2 Treatment-Reversed New-Onset Diabetes in Nonobese Diabetic Mice Am. J. Pathol., April 1, 2008; 172(4): 972 - 979. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. You, B. Leforban, C. Garcia, J.-F. Bach, J. A. Bluestone, and L. Chatenoud Adaptive TGF-beta-dependent regulatory T cells control autoimmune diabetes and are a privileged target of anti-CD3 antibody treatment PNAS, April 10, 2007; 104(15): 6335 - 6340. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Tian, D. Zekzer, Y. Lu, H. Dang, and D. L. Kaufman B Cells Are Crucial for Determinant Spreading of T Cell Autoimmunity among beta Cell Antigens in Diabetes-Prone Nonobese Diabetic Mice J. Immunol., February 15, 2006; 176(4): 2654 - 2661. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. D. Dai, K. P. Jensen, A. Lehuen, E. L. Masteller, J. A. Bluestone, D. B. Wilson, and E. E. Sercarz A Peptide of Glutamic Acid Decarboxylase 65 Can Recruit and Expand a Diabetogenic T Cell Clone, BDC2.5, in the Pancreas J. Immunol., September 15, 2005; 175(6): 3621 - 3627. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Han, Y. Li, J. Wang, R. Wang, G. Chen, L. Song, R. Xu, M. Yu, X. Wu, J. Qian, et al. Active Tolerance Induction and Prevention of Autoimmune Diabetes by Immunogene Therapy Using Recombinant Adenoassociated Virus Expressing Glutamic Acid Decarboxylase 65 Peptide GAD500-585 J. Immunol., April 15, 2005; 174(8): 4516 - 4524. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. You, C. Chen, W.-H. Lee, T. Brusko, M. Atkinson, and C.-P. Liu Presence of Diabetes-Inhibiting, Glutamic Acid Decarboxylase-Specific, IL-10-Dependent, Regulatory T Cells in Naive Nonobese Diabetic Mice J. Immunol., December 1, 2004; 173(11): 6777 - 6785. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-K. Kim, K. V. Tarbell, M. Sanna, M. Vadeboncoeur, T. Warganich, M. Lee, M. Davis, and H. O. McDevitt Prevention of type I diabetes transfer by glutamic acid decarboxylase 65 peptide 206-220-specific T cells PNAS, September 28, 2004; 101(39): 14204 - 14209. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Chen, W.-H. Lee, P. Yun, P. Snow, and C.-P. Liu Induction of Autoantigen-Specific Th2 and Tr1 Regulatory T Cells and Modulation of Autoimmune Diabetes J. Immunol., July 15, 2003; 171(2): 733 - 744. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Tian, A. P. Olcott, and D. L. Kaufman Antigen-Based Immunotherapy Drives the Precocious Development of Autoimmunity J. Immunol., December 1, 2002; 169(11): 6564 - 6569. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. J. Quintana, P. Carmi, and I. R. Cohen DNA Vaccination with Heat Shock Protein 60 Inhibits Cyclophosphamide-Accelerated Diabetes J. Immunol., November 15, 2002; 169(10): 6030 - 6035. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. V. Tarbell, M. Lee, E. Ranheim, C. C. Chao, M. Sanna, S.-K. Kim, P. Dickie, L. Teyton, M. Davis, and H. McDevitt CD4+ T Cells from Glutamic Acid Decarboxylase (GAD)65-specific T Cell Receptor Transgenic Mice Are Not Diabetogenic and Can Delay Diabetes Transfer J. Exp. Med., August 19, 2002; 196(4): 481 - 492. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. H. Friedline, C. P. Wong, D. A. Steeber, T. F. Tedder, and R. Tisch L-Selectin Is Not Required for T Cell-Mediated Autoimmune Diabetes J. Immunol., March 15, 2002; 168(6): 2659 - 2666. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Kim, M. Zhang, B. P. Vistica, C.-C. Chan, D.-F. Shen, E. F. Wawrousek, and I. Gery Induction of Ocular Inflammation by T-Helper Lymphocytes Type 2 Invest. Ophthalmol. Vis. Sci., March 1, 2002; 43(3): 758 - 765. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
